Abstract:The RNA-guided endonuclease Cas9 from microbial immune systems is a powerful tool for genome editing in eukaryotic cells. The nuclease activity of Cas9 can be triggered when there is imperfect complementarity between the RNA guide and target DNA. This is a problem in the context of genetic therapies, where one would like precise specificity. Crystal structures of bound and unbound Cas9 have revealed substantial information on CRISPR/Cas9 systems. However, resolved crystal structures have two major limitations: (i) the nuclease active site is mostly far from the target DNA (DNAt) strand cleavage site and (ii) there is lack of an intact non-target DNA (DNAnt) strand.
Here I focus on the latter limitation. Using a working hypothesis formulated by Slaymaker and coworkers , based on the existence of a positively charged patch in Cas9 that ideally accommodates the negatively charged backbone of the unwound DNAnt strand, we modeled a longer DNAnt strand in the crystal structure of the ternary complex from PDB ID 4UN3. The molecular dynamics (MD) of this complex on the scale of 1.5 μs, complemented by electron paramagnetic resonance (EPR) measurements of distances between spin labels attached to the backbone of DNAt and DNAnt, offers consolidation to this working hypothesis2 .
I present an overview of CRISPR/Cas9 operation and the results of the joint MD-EPR work2 on DNAt-DNAnt distances, with implications on detecting the fate of the unwound DNAnt strand after binding of the RNA and endonuclease. Furthermore, I outline current ongoing efforts, preliminary results and future plans to unravel possible structural ways to improve the binding specificity of CRISPR/Cas9 complexes through mutations in the above mentioned positively charged patch1.